Engineering bacteria for expressing benzene ring bioxygerase and its application

A technology of benzene ring dioxygenase and chlorobenzene dioxygenase, which can be applied in application, genetic engineering, bacteria and other directions, can solve problems such as low background expression, and achieve broad application prospects, simple production process and transformation efficiency. high effect

Inactive Publication Date: 2006-11-29
TSINGHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Environmental Microbiology, 2002, 4(12), 824-841), but because its background expression level is usually low, wild-type Pseudomonas can not be used as the production bacteria that converts benzene into DHCD

Method used

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  • Engineering bacteria for expressing benzene ring bioxygerase and its application
  • Engineering bacteria for expressing benzene ring bioxygerase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048]Embodiment 1, the construction of special expression engineering bacteria of phenyl ring dioxygenase

[0049] One, the construction of the Pseudomonas expression vector containing toluene dioxygenase gene (tod)

[0050] 1) Plasmid pKST11 (Allen, C.C.R., Boyd, D.R., Dalton, H., et.al.Sulfoxides of high enantiopurity from bacterial dioxygenase-catalysed oxidation.J.Chem.Soc. Chem.Comm.1995, 119-120) was subjected to double enzyme digestion, and the reclaimed toluene dioxygenase gene fragment (NCBIGenBank number is TodC1, TodC2, TodB and TodA genes described in J04996) with a size of 3866bp;

[0051] 2) Digest the plasmid pBBR1MCS-2 (NCBI GenBank No. U23751) with the restriction endonuclease BamHI, and recover the fragments;

[0052] 3) Ligate the recovered fragments in step 1) and step 2) with T4 DNA ligase. After the ligation reaction is completed, introduce the ligation product into E.coli JM109 (ATCC number: 53323) by electrotransformation, and convert the Spread the ...

Embodiment 2

[0074] Example 2, Determination of Enzyme Activity of Benzene Dioxygenase Expressed by Benzene Ring Dioxygenase Special Expression Engineering Bacteria

[0075] Detect the enzymatic activity of the benzene ring dioxygenase expressed by four kinds of benzene ring dioxygenase special-purpose expression engineering bacteria that embodiment 1 constructs with following method, concrete method is as follows:

[0076] Inoculate Pseudomonas putida KT2442 (pSPM01), Pseudomonas stutzeri 1317 (pSPM01), Pseudomonas putida KT2440 (pSPM01) and Pseudomonas aeruginosa PAO1 (pSPM01-1) in LB-Km liquid medium, respectively. , cultivated on a shaking table at 30°C and 200rpm for 12h, then inoculated the four bacterial liquids into LBG-Km liquid medium at a ratio of 5% (V / V), cultured on a shaking table at 30°C and 200rpm, respectively At different incubation times (4, 8, 12, 16, 24, 36, 48h), 2 mL samples were taken to detect the enzyme activity of benzene ring dioxygenase. The detection method ...

Embodiment 3

[0078] Embodiment 3, use Pseudomonas stutzeri 1317 (pSPM01) to convert benzene to produce the shake flask experiment of DHCD

[0079] 1) Inoculate Pseudomonas stutzeri 1317 (pSPM01) in LB-Km liquid medium, culture on a shaking table at 35°C and 200 rpm for 12 hours, and then inoculate the bacterial liquid into LBG-Km liquid at a ratio of 5% (V / V) In the culture medium, culture on a shaker at 35°C and 200rpm for 12h;

[0080] 2) Dissolving benzene in soybean oil to form a benzene solution, the volume ratios of benzene and soybean oil are 1:1 and 1:3 respectively;

[0081] 3) benzene and the benzene solution prepared in step 2) were added to step 1) respectively in the fermented liquid containing Pseudomonasstutzeri 1317 (pSPM01), so that the concentration of benzene in the fermented liquid was respectively 5, 10, 15 mL / L, and then at 35 Continue to cultivate on a shaking table at 200 rpm for 8 hours, and use Bio-RAD Company (U.S.) Biologic Duo-Flow HPLC system (the chromatogra...

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Abstract

The invention discloses an engineering fungus, which could express benzene ring dioxygenase, and the application. Especially, it involves the special expressing engineering fungus of benzene ring dioxygenase and the application in producing syn-1, 2-dihybric-3, 5-cyclohexane. The engineering fungus is gained by inducing the expressing carrier containing benzene ring dioxygenase gene into fake single cell fungus. The producing method of syn-3, 5-dihyadric-1, 2-cyclohexadiene contains the following steps: fermenting the engineering fungus to gain benzene ring dioxygenase; adding substrate benzene or benzene solution, and the content adding to fermenting liquid is 10-30mL/L every hour; fermenting for 8-24 hours to gain syn-1, 2-dihybric-3, 5-cyclohexane; solvent of the benzene solution could be olefin or bean oil, and the volume ratio of benzene and solvent is 1:1-3. The special expressing engineering fungus of benzene ring dioxygenase will perform an important function in the industrial producing of DHCD, and it has great application prospect.

Description

technical field [0001] The present invention relates to a special expression engineering bacterium of benzene ring dioxygenase and its application, in particular to a kind of special expression engineering bacterium of benzene ring dioxygenase and the engineering bacterium used in the production of cis-1,2-dihydroxy-3 , the application of 5-cyclohexadiene. Background technique [0002] Cis-1,2-dihydroxy-3,5-cyclohexadiene (DHCD, see formula I for chemical structural formula) is chemically synthesized high-performance material polyphenylene (see formula II for chemical structural formula, n=1,2,3,4 ,…) monomer. Polyphenylene is a high-performance polymer with a series of excellent properties such as good stability, high strength, high temperature resistance, flame retardancy, good durability, corrosion resistance, and good electrical properties. Polyphenylene has potential application value in aerospace, nuclear energy, electronic information, precision machinery, petrochem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/53C12P7/02C12P1/04C12R1/38
Inventor 陈国强欧阳少平孙尚瑜陈金春
Owner TSINGHUA UNIV
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